The present invention comprises an improved family of bulk starter media and the use of these media for propagation of useful acid producing bacteria. The products and process of the invention are particularly beneficial in producing bacterial cultures for fermentation processes in the food industry.
Many important food products are prepared by deliberate controlled bacterial fermentation. Among these can be mentioned the many products of milk fermentation such as hard and soft cheeses, cottage cheese, and yogurt. Many types of pickles and sausages are also dependent upon bacterial fermentation for proper texture and flavor development. Cheddar cheese production can be cited as exemplary of the many food fermentation processes. In its commercial production, large vats of milk are treated with an enzyme, such as rennett, in order to induce clotting. Additionally, the milk is inoculated with cultures of lactic acid producing bacteria to provide the required acidic conditions, as well as the desired flavor, texture, and odor characteristics of the cheese. These bacteria act mainly upon lactose and similar carbohydrate materials and produce increasing quantities of lactic acid as the fermentation proceeds. While there are a great many species and strains of lactic acid producing bacteria, relatively few have assumed commercial importance. Among these can be included Streptococcus cremoris, S. lactis, S. thermophilus, S. diacetylactis, and Lactobacillus bulgaricus.
The bacterial cultures which are added to the milk are generally propagated at the dairy or cheese plant from selected pure strains of the bacteria available commercially from a number of sources well known within the industry. These starter bacteria are normally sold and maintained in frozen condition so that their activity can be preserved for long periods of time. When required for use, the starter bacteria are added to a growth medium, generally called a bulk starter medium, which contains essential mineral and organic nutrients which are known from experience to be required for the particular species being propagated. In particular, the bulk starter must contain energy materials, usually sugars and similar readily metabolized carbohydrates, and a suitable source of nitrogen. Very often a source of phosphorus is also required. Growth media are normally supplied as a dry granular mixture. This is dissolved or suspended in water and pasteurized to destroy any ambient pathogens. It is then cooled and inoculated with the bacteria to be propagated. The culture thus produced is maintained at slightly elevated temperature for a period of time sufficient to allow the growth of about 10.sup.6 to 10.sup.8 colony forming units or cells (cfu) per ml. In many cheese plants, it is convenient to incubate the starter culture overnight. This will then be used the next morning for cheese production.
One problem which frequently besets bacterial fermentations is infection with bacteriophages. Each of the major strains of cheese producing bacteria also appears to have a specific bacteriophage (or simply phage) which has evolved as a homologous antagonist. In response to this problem, fcod scientists have developed culture or starter media which are phage-inhibitory. Gulstrom and coworkers, J. Dairy Sci., 62:208-221 (1979), have evaluated eight commercially available phage-inhibitory starter media using a large number of different bacterial strains and phages. The most effective of the group of media evaluated appear to be those having phosphate-citrate buffer systems. Gulstrom et al. point out that available calcium ion is an essential for phage propagation. A response to this knowledge has been the formulation of low calcium starter media, or media which contain materials such as orthophosphate salts or other materials which bind calcium and make it unavailable. Citrate buffers are also known to be effective.
Unfortunately, many media which offer good phage control are not particularly good for nourishing the desired bacteria. Gulstrom et al. point out that in a medium, the ability to support good bacteria growth and at the same time suppress phage proliferation are mutually exclusive properties. Some optimum balance must be sought.
Another ingredient often added to lactic acid starter media is a buffering salt. The initial pH of media for propagation of cheese Streptococcal bacteria is normally in the range of 6.5-6.8. As the bacteria proliferate, the pH drops due to the generation of lactic acid. Below a pH of about 5, bacterial growth slows markedly and the bacteria begin to lose vigor. In actuality, they are being killed or deactivated by the acid they themselves have generated. One widely used commercial system employs a continuous addition of ammonia which serves to neutralize the acid as it is generated. Other systems employ sparingly soluble magnesium compounds, such as magnesium phosphate or magnesium ammonium phosphate, which are solubilized as the pH drops and serve to neutralize a portion of the acid. U.S. Pat. Nos. 4,282,255 and 4,382,965 to Sandine et al. disclose phage-inhibitory media containing magnesium phosphate and/or magnesium ammonium phosphate for acid neutralization. Additionally, they disclose the use of microencapsulated alkaline materials, such as sodium carbonate, which dissolve slowly and serve to give a controlled time release of alkali to the system for acid neutralization. Sinkoff et al., U.S. Pat. No. 4,402,986, describe an aqueous bulk starter medium containing magnesium ammonium phosphate hexahydrate as a neutralizing agent.
One of the problems of starter media containing insoluble or sparingly soluble inorganic compounds is the fact that many of them require agitation during the entire incubation time of the starter media in order to maintain these materials in suspension. Though this requirement for constant agitation is sometimes regarded as a nuisance, it can also be undesirable if it causes excessive oxygenation of the culture. Excessive oxygen tends to retard and inhibit bacterial growth.
Some workers have found that particular metal ions in trace quantities enhance bacterial growth. Etchells et al., U.S. Pat. No. 3,410,755, and Canadian Pat. No. 1,024,393 each disclose the addition of small quantities of magnesium, manganese, and ferrous ions in bacterial growth media.
Despite the commercial availability of several excellent bulk starter media, none to date has proved ideal for the cheesemaker. Continuous ammonia addition requires exceedingly careful attention and control. Some media which have shown excellent properties of phage inhibition often produce cultures of low activity. The present inventors have recognized these problems and have now developed a family of bulk starter media which overcome most of them. The media produced by the teachings of the present invention are phage-inhibitory, do not require continuous agitation, and are internally buffered so that bacterial growth is not inihibited by excessive acid production.